We have been investigating the energetics and mechanisms of binding protein-dependent active transport in E. coli, using glutamine transport system as a model. We have succeeded in reconstituting this transport system in spheroplasts and established that glutamine transport requires glutamine binding protein and at least one other macromolecular component. We have also obtained several glutamine transport deletion mutants and using these deletions we have cloned glutamine transport system onto the plasmid pBR322. The cloned DNA is 3.7 Kbp long. Work is now in progress in subcloning and restriction site mapping. Phosphoenolpyruvate transport system of Salmonella typhimurium has been cloned in the plasmid pBR322. With this clone E. coli which is devoid of this transport system can now transport PEP and grow on it as a sole source of carbon energy. In collaboration with H.R. Kaback of the Roche Institute of Molecular Biology we are using this clone as a means by which to synthesize ATP inside the membrane vesicles to study other proton-motive force-driven transport systems. Attempt will be made to identify the PEP transport protein encoded on the clone using minicell preparation and in vitro as well. The DNA sequence of this transport system will be determined.